Shikimic acid (trihydroxy-1-cyclohexene-1-carboxylic acid; Chemical Abstracts Registry Number 138-59-0) is the key precursor compound for the synthetic manufacture of oseltamivir ((3R,4R,5S)-4-(acetyl-amino)-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid ethyl ester; Chemical Abstracts Registry Number 196618-13-0) (Rohloff et al., 1998; Federspiel et al., 1999). Oseltamivir, an orally active inhibitor of the essential neuraminidase of influenza virus, was discovered by scientists at Gilead Sciences Inc. of Foster City, Calif. (Kim et al., 1997; Kim et al., 1998; Abrecht et al., 2004; Bischofberger et al., U.S. Pat. No. 5,763,483; Lew et al., U.S. Pat. No. 5,866,601; Bischofberger et al., U.S. Pat. No. 5,952,375; Bischofberger et al., European Patent EP 0759917 B1; Bischofberger et al., European Patent EP 0976734 B1).
During influenza virus replication, new virus particles are bound to a sialic acid side-chain on the virus receptor protein. The mechanism of the viral neuraminidase is to cleave off this sialic acid and release the newly replicated virus particles. Oseltamivir is a sialic acid analog that inhibits this cleavage reaction by binding to the active site of the neuraminidase. These abortively infected cells are destroyed, stopping the spread of the virus within the host. Oseltamivir has potential use in influenza pandemics, including of “bird flu”, in the form of the pharmaceutical TAMIFLU® (De Clercq, 2002; Bradley, 2005; Farina and Brown, 2006). The pharmaceutical TAMIFLU® is the phosphate salt of oseltamivir (Chemical Abstracts Registry Number 204255-11-8; also known as Roche compound Ro 64-0796/002 and Gilead Sciences compound GS-4104-02). TAMIFLU® was first marketed by Roche in October 1999 (Farina and Brown, 2006). However, the large-scale production of the drug has been limited by the low availability of the shikimic acid precursor material.
Shikimic acid is a scarce and expensive chemical substance, being obtained principally from the seeds of woody shrubs, namely the Chinese star anise shrub (Illicium verum) native to China, and the shikimi-no-ki shrub (Illicium anisatum, formerly called Illicium religiosum) from whence shikimic acid got its name, native to Japan (Haslam, 1974; Sadaka and Garcia, 1999; Payne and Edmonds, 2005). About 30 kilograms of star anise or shikimi-no-ki seeds are required to produce one kilogram of shikimic acid (Farina and Brown, 2006). However, this natural source is limited, and insufficient to meet worldwide demand for TAMIFLU® (Bradley, 2005).
1.3 grams of shikimic acid are required to manufacture the 10 doses of TAMIFLU® needed to treat one person (Bradley, 2005). To produce enough TAMIFLU® to treat 400 million people (a conservative estimate of the need in the event of an influenza pandemic) would require 520,000 kilograms of shikimic acid. Worldwide annual production of shikimic acid is currently only about 100,000 kilograms. Another estimate of the need for TAMIFLU® in the event of a severe influenza pandemic is 30 billion doses, requiring 39 million kilograms of shikimic acid (Bradley, 2005).
Two alternative approaches to resolving the problem of shikimic acid scarcity have been recently explored. The first is the production of shikimic acid by microorganisms by a fermentation-based process (Farina and Brown, 2006). The second is based on new chemical synthesis routes to oseltamivir phosphate that do not utilize scarce natural products as precursor compounds, but rather use inexpensive and widely available chemicals (Fukuta et al., 2006; Yeung et al., 2006). The chemical routes that have been developed to date are functional only as academic, bench-scale syntheses, and are not efficient industrial processes that could compete with the current shikimic acid-based manufacturing process for oseltamivir phosphate (Farina and Brown, 2006).
To understand the fermentation-based processes, it would be useful to briefly review the biosynthetic pathway to shikimic acid. This pathway is known both as the common aromatic biosynthetic pathway (Herrmann, 1983; Pittard, 1987; Pittard, 1996) because it leads to (among other things) the aromatic amino acids, and also as the shikimate pathway (Haslam, 1974) after the metabolic intermediate in the pathway that was identified first. Several entire books and comprehensive review articles have been devoted to this important metabolic pathway (Haslam, 1974; Weiss and Edwards, 1980; Herrmann, 1983; Conn, 1986; Pittard, 1987; Haslam, 1993; Herrmann, 1995a; Herrmann, 1995b; Pittard, 1996; Herrmann and Weaver, 1999; Bongaerts et al., 2001; Kramer et al., 2003).
The common aromatic biosynthetic pathway is present in plants, bacteria, fungi, and other eukaryotic microorganisms. A search of on-line databases by the Applicants, specifically PubMed and the National Center for Biotechnology Information (NCBI), indicated that in addition to plants, bacteria, and fungi, the pathway is present in Stramenopiles such as brown algae and diatoms, Alveolata (within the Protista kingdom) such as ciliates, dinoflagellates and apicomplexa parasites, and various Euglenozoa. The common aromatic biosynthetic pathway is not found in most higher animals, such as nematodes, insects and other arthropods, mollusks, and vertebrates and other chordates including fishes, amphibians, reptiles, birds and mammals. It has been established by others that the common aromatic biosynthetic pathway is present in the parasitic protozoan microorganisms known as apicomplexa (Roberts et al., 1998; McConkey, 1999; Roberts et al., 2002). It has also been suggested by others that the common aromatic biosynthetic pathway may be present in some higher animals, specifically in basal metazoans among the marine and freshwater invertebrates known as cnidarians (or coelenterates), including corals, sea anemones, jellyfishes, and hydroids (Starcevic et al., 2008). It is to be understood that statements that the common aromatic biosynthetic pathway is present in microorganisms mean that the pathway occurs in microscopic organisms and taxonomically related macroscopic organisms within the categories algae, Archaea, bacteria, fungi, and protozoa; this includes prokaryotes, including cyanobacteria, as well as unicellular eukaryotic organisms.
The facts that microorganisms possess the common aromatic biosynthetic pathway, and depend on it for the biosynthesis of many essential cellular components, and that mammals (including humans) lack the pathway, make the enzymes of the pathway attractive targets for new classes of antimicrobial therapeutic agents (Davies et al., 1994; Roberts et al., 1998). Any such therapeutic agents that are based on shikimic acid would increase the demand for shikimic acid. Indeed, 6-fluoroshikimic acid has been found to be an effective anti-bacterial compound (Davies et al., 1994; Bornemann et al., 1995) and anti-parasitical compound (McConkey, 1999; Roberts et al., 2002). Shikimic acid has also been converted into compounds that exhibited a significant inhibitory effect on cell proliferation, opening their possible use as anti-cancer chemotherapeutic agents (Tan et al., 1999). Thus, shikimic acid could serve as an important building block for a wide array of important classes of drugs, including anti-viral, anti-bacterial, anti-parasitical, and anti-cancer drugs.
In the bacterium Escherichia coli, the first step in the common aromatic biosynthetic pathway (FIG. 1) is carried out by three isofunctional DAHP synthase enzymes; these three isofunctional enzymes are encoded by the aroF, aroG, and aroH genes. Similarly, there are two isofunctional enzymes of shikimate kinase, encoded by the aroK and aroL genes. The other enzymes of the pathway consist of single enzymes and are encoded by single genes: the aroB gene encodes 3-dehydroquinate synthase, the aroD gene encodes 3-dehydroquinate dehydratase, the aroE gene encodes shikimate dehydrogenase, the aroA gene encodes EPSP synthase, and the aroC gene encodes chorismate synthase.
A fermentation-based route to shikimic acid was described by John W. Frost and co-workers at Michigan State University who reported construction of a strain of E. coli with several genetic modifications that led to the production of shikimic acid (Draths et al., 1999; Frost et al., International Publication No. WO 00/44923; Frost et al., International Publication No. WO 02/29078; Frost et al., U.S. Pat. No. 6,472,169; Frost et al., U.S. Pat. No. 6,613,552). These modifications included adding a second copy of the aroB gene, encoding 3-dehydroquinate synthase, to the chromosome of the host cell. In addition, a mutant form of the aroF gene, encoding a feedback-resistant DAHP synthase enzyme, and a wild-type copy of the aroE gene, encoding shikimate dehydrogenase, were placed on a multicopy plasmid in the host cell. These genetic modifications increased the flow of metabolites to shikimic acid. Finally, these workers described genetically disabling (“knocking out”) the aroK and aroL genes encoding the two isofunctional shikimate kinase enzymes. Such a disabled strain would be, in theory, completely blocked in the conversion of shikimic acid to shikimate-3-phosphate (FIG. 1) and would accumulate shikimic acid.
Early on, it was reported that a mutant with such a block in the pathway would accumulate shikimic acid (Davis and Mingioli, 1953). However, such a disabled strain would also be unable to make the aromatic amino acids tryptophan, phenylalanine, and tyrosine, as well as the other essential compounds para-hydroxybenzoic acid, para-aminobenzoic acid, and 2,3-dihydroxybenzoic acid (FIG. 1). Indeed, in order to grow this strain in a fermenter, the fermentation medium had to be supplemented with all six of these essential compounds, specifically tryptophan (350 milligrams per liter), phenylalanine (700 milligrams per liter), tyrosine (700 milligrams per liter), para-hydroxybenzoic acid (10 milligrams per liter), para-aminobenzoic acid (10 milligrams per liter), and 2,3-dihydroxybenzoic acid (10 milligrams per liter) (see, for example, Draths et al., 1999, in supplementary materials published on-line; Frost et al., International Publication No. WO 00/44923, page 13, lines 24-30; Frost et al., International Publication No. WO 02/29078, page 28, lines 5-11; Frost et al., U.S. Pat. No. 6,472,169, column 16, lines 23-33; and Frost et al., U.S. Pat. No. 6,613,552, column 10, lines 23-33). The levels of shikimic acid in fermentation cultures of this strain ranged from 20 to 50 grams per liter. Further work with this strain by Frost and colleagues has improved the yield of shikimic acid to up to 80-90 grams per liter (Knop et al., 2001; Bongaerts et al., 2001; Chandran et al., 2003; Kramer et al., 2003). A nearly identical approach has been reported by workers in Sweden (Johansson et al., 2005; Johansson and Liden, 2006; Johansson, 2006).
A similar approach was reported by workers in Russia (Iomantas et al., U.S. Pat. No. 6,436,664) who used mutant strains of the bacterial genus Bacillus that were deficient in shikimate kinase to produce shikimic acid by fermentation. The fermentation medium, as necessitated by such mutants, had to be supplemented with the required compounds (see column 5, lines 38-52 of the patent). The levels of shikimic acid in fermentation cultures of these strains ranged from 3 to 17 grams per liter. Workers in Japan (Shirai et al., European Patent Application EP 1092766 A1) reported a similar approach with mutant strains of the bacterial genus Citrobacter to produce shikimic acid by fermentation. Again, the fermentation medium had to be supplemented with the required compounds (see, for example, Tables 3 and 4 of the patent application). The levels of shikimic acid in fermentation cultures of these strains ranged from 4 to 10 grams per liter. These various approaches have been the subject of review articles by Bongaerts et al. (2001) and Kramer et al. (2003). Methods have been developed for the recovery of shikimic acid from fermentation cultures (Malmberg and Westrup, U.S. Pat. No. 6,794,164; Van der Does et al., International Publication No. WO 02/06203).
All previous fermentation-based processes for the production of shikimic acid discussed here employ fermentation culture media containing expensive nutrient components such as tryptophan, phenylalanine, tyrosine, para-hydroxybenzoic acid, para-aminobenzoic acid, and 2,3-dihydroxybenzoic acid, or other expensive nutrient components. The cost of these nutrient component additions is considerable. The addition of tryptophan, phenylalanine, tyrosine, para-hydroxybenzoic acid, para-aminobenzoic acid, and 2,3-dihydroxybenzoic acid at the levels disclosed in the papers, patents, and patent applications discussed above (Draths et al., 1999; Frost et al., International Publication No. WO 00/44923; Frost et al., International Publication No. WO 02/29078; Frost et al., U.S. Pat. No. 6,472,169; Frost et al., U.S. Pat. No. 6,613,552) would increase the cost of the culture medium by about one dollar per liter (in 2007 US dollars).
Additionally to produce 39 million kilograms of shikimic acid per year by a fermentation process yielding 80 grams of shikimic acid per liter, and also requiring the amounts of the nutrient components reported in the cited papers, patents, and patent applications, would require (each year) about 340 metric tons each of tyrosine and phenylalanine, about 170 metric tons of tryptophan, and about 490 kilograms each of para-hydroxybenzoic acid, para-aminobenzoic acid, and 2,3-dihydroxybenzoic acid. As one example of the limitations imposed by the worldwide supply of these nutrients, the entire yearly global production of tyrosine is about 120 metric tons per year (Ikeda, 2003), far short of the 340 metric tons of tyrosine needed to produce 39 million kilograms of shikimic acid by the cited fermentation processes. In contrast, the cost of using the chemically defined minimal culture medium described in the instant invention (including the addition of glyphosate) would cost about 100-fold less, and at the most would consume about 3700 metric tons of glyphosate, which is only about 0.8% of the yearly global production of about 460,000 metric tons of glyphosate (N-(phosphonomethyl)-glycine; Chemical Abstracts Registry Number 1071-83-6). Glyphosate, inter alia, is an inhibitor of the enzyme 5-enolpyruvoylshikimate-3-phosphate synthase (EPSP synthase), and it therefore inhibits the common aromatic biosynthetic pathway at the point of conversion of shikimate-3-phosphate by the enzyme EPSP synthase to EPSP (FIG. 1). Inhibition of EPSP synthase by glyphosate may lead to the accumulation of shikimate-3-phosphate in the cell.
Anderson et al. (2001) disclose a method for the determination of shikimic acid in plant tissue after exposure of the plant to glyphosate. Shikimic acid analysis of the plant tissue was performed using water extraction followed by high-pressure liquid chromatography (HPLC) analysis.